The overall goal of this application is to investigate the pathogenesis of prion diseases using transgenic (Tg) mouse models. Our objective is to understand the molecular and cellular mechanisms by which prions kill neurons and damage the CNS. A major focus of our work has been on Tg mice that express a PrP molecule with a nine-octapeptide insertional mutation (PG14) associated with a familial form of Creutzfeldt- Jakob disease in humans. These mice model several essential features of inherited human prion diseases, including progressive ataxia, neuronal loss, astrocytosis, and accumulation of an abnormally folded form of mutant PrP. During the previous funding period, we created new lines of Tg mice which selectively express PG14 PrP in forebrain neurons under control of a tetracycline-regulated promoter; we investigated whether ER-associated degradation plays a role in the metabolism of the mutant protein; and we compared the molecular and biological properties of two forms of PG14 PrP that differ dramatically in their infectivity and oligomeric structure. In the present application, we propose to expand our search for PrP-related pathogenic processes. In each of the aims, we will explore one of three complementary mechanisms by which PrP might produce neurotoxic effects: gain of function, loss of function, and subversion of function. In the first aim, we will use Tg mice expressing a GFP-tagged version of PG14 PrP to explore the hypothesis that aggregates of PG14 PrP interfere with axonal transport and synaptic function, and that this toxic activity contributes to the disease phenotype in Tg(PG14) mice. In the second aim, we will determine whether loss of a normal neuroprotective activity of PrP contributes to neurodegeneration in Tg(PG14) mice. In the third aim, we will investigate the neurotoxic effects of a deleted form of PrP that may act by subverting the normal function of PrP, much like PrPSc is thought to do during prion infection. We anticipate that these studies will provide insights into the process of prion-induced neurodegeneration, and will identify molecular targets for therapeutic intervention in these and other neurodegenerative disorders.